Beyond Dmax and DC50: A simple tool to evaluate the complex PROTAC-mediated kinetic degradation dynamics

Protein degradation mediated by PROteolysis TArgeting Chimeras (PROTACs) is a complex, dynamic, and multi-step process. The structural diversity of PROTACs, coupled with cellular heterogeneity, introduces considerable variability at each stage. Therefore, a straightforward yet comprehensive approach is essential to effectively dissect these mechanisms. The existing methods for evaluating PROTAC degradation are often time-consuming, labor-intensive, or expensive, and many capture only a partial view of the entire degradation process. Consequently, multiple complementary assays are typically required to obtain the complete picture. Here, we use commercially available Cyclin dependent Kinase 6 (CDK6) targeting PROTACs to demonstrate that a simple, single-step, no-wash, additive-free, GFP-based live-cell imaging approach can rapidly generate dynamic degradation metrics (Dmax, Rmax, TDmax, CDmax, DC50, Km, Tmax, Kdeg, Vmax, DT50, {tau} and TRmax) for comparing and ranking PROTACs within a controlled reporter system. The rapid assay turnaround time, excellent assay qualities and compatibility with high-throughput (HT) formats will facilitate quick hit identification and serve as a guiding assay for hit-to-lead optimization campaigns.